JP3502125B2 - Extraction and culture of transformed cells and production of antibodies against them - Google Patents

Abstract

A process with which it is possible in a non-hazardous way without the addition of chemicals or other aids to isolate from a living individual transformed cells and individual-specific polyclonal or monoclonal antibodies which are directed against tumour-specific antigens on these transformed cells for diagnostic and therapeutic purposes is described.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for extracting transformed cells from the bloodstream of a human or animal individual, a method for culturing cells obtained by such a method, and a monoclonal antibody to those cells. Related to the manufacture of.

[0002]

The importance of transformed cells circulating in the bloodstream to form metastases is described by AJ Salsbury in "Sign.
ificance of Circulating Cancer Cells "(William He
inemann Medical Books, New Aspects of Breast Canse
r, Vol. 3, pages 245 et seq., 1977), these cancer cells circulating in the bloodstream are released, for example, during a period of up to 5-6 hours after being released by a particular surgical treatment modality. Reports that they are unable to form metastases. The presence of large numbers of cancer cells is not sufficient for the appearance of metastases and the cancer cells would first have to leave the ductal area. Nevertheless, on the one hand, it impedes the formation of thrombin from the bloodstream as a starting point for metastasis formation,
On the other hand, the use of agents to kill transformed cells has been proposed.

However, more recent knowledge (PM Gullino,
Dev. Oncol., 49, 251-271 (1986)) found that circulating tumor cells often appear in the bloodstream long before clinical evidence of the primary tumor is possible. Reveals. Although not all transformed cells are capable of inducing metastasis, it is necessary to stop the growth of circulating tumor cells and thus the growth of metastases as early as possible. The reason why a clear correlation between the number of transformed cells in the peripheral blood and the induction of metastasis formation has not been established up to now is, to a large extent, the demonstration of transformed cells in the bloodstream. Because it is difficult.

Furthermore, when using conventional diagnostic methods such as palpation of solid tumors, there is a marked increase in the release of transformed tumor cells, especially in the case of biopsy and surgical treatments. It is known that this can lead to increased metastasis formation.

The behavior of solid tumors in the formation of metastases via the lymphatic and / or circulatory system has, up to now, only been studied in vivo in open blood vessels during surgery. Circulating tumor cells were not obtained in diagnostically or therapeutically useful amounts. Therefore, the behavior of solid tumors in forming metastases in the circulatory system could only be studied in animal experiments or in autopsies of humans by pathologists.

There are many known methods for extracting transformed cells from blood. For example, chemical or enzymatic lysis of non-transformed cells, accelerated erythrocyte sedimentation, flotation, centrifugation and magnetic separation. JA Fleming and J.
Review by W. Stewart, Journal of Clinical Patholo
gy, 20, 145-151 (1967) and JT West and R.
Progress in Clinical Pathology, Vol. By H. Hume
3, Chap. 11, 362-382 (1970). These known methods usually lead to low yields and in particular present the drawback that it is not possible to determine whether the transformed cells obtained from the peripheral circulation were alive at the time of extraction. Nevertheless, I have relied on these methods to this day.

Therefore, transformed cells are, for example, Fico
It can be extracted by density gradient centrifugation as is commonly used for isolation of lymphocytes in ll® silicon, ie an albumin gradient. However, this requires a large amount of blood, which places a heavy burden on the patient. Moreover, despite the high cost of the device, the yields based on a small number of transformed cells are very low and, moreover, these transformed cells are susceptible to damage and impure by conventional centrifugation methods. Therefore, density gradient centrifugation cannot be used as a conventional method to extract large numbers of transformed cells. Methods of recirculating other blood components to the patient, such as plasma and red blood cells, cannot be performed due to impurities created by the gradient material. Centrifugation is not suitable for clinical application to patients.

Thus, to date, transformed metastatic cells, presumably circulating in the peripheral circulation, have been extracted by non-surgical means, cultivated them in vitro and used for further investigations and other uses. There was no reproducible and convenient way to do this.

[0009]

DISCLOSURE OF THE INVENTION The present invention makes it possible to utilize a method capable of extracting transformed cells circulating in the blood stream obtained from a human or animal individual by a simple and safe means. It is based on what you want to do.

In this connection, the cells obtained by such a method are cultured and replicated to produce a monoclonal antibody against the antigen on the surface of such a cell, which is used for diagnosis and / or therapy. The purpose is to be able to use for the purpose of.

[0011]

SUMMARY OF THE INVENTION The present invention provides that when whole blood is separated in an apheretic device suitable for this purpose, it will, under certain circumstances, produce a fraction that also contains leukocytes and / or lymphocytes. It is based on the recognition that it is rich in living transformed cells.

Thus, the purpose is to obtain a large amount of transformed cells from the blood by means of a phlebotomy device suitable for separating the blood into its constituents, and under certain circumstances leukocytes and / or lymphocytes. It is achieved by a method of isolating and culturing a transformed cell circulating in the bloodstream, in that the fraction containing also is recovered and the transformed cell is replicated.

To produce individual-specific antibodies to tumor antigens, cultured transformed cells are contacted with B lymphocytes to stimulate the production of antibodies to tumor antigens on the transformed cells, such as The antibody is immortalized, allowed to replicate, and the stimulated B lymphocytes are selected, the antibody is isolated from the immortalized B lymphocyte culture, and such antibody is purified.
The antibody thus obtained may be polyclonal, but is preferably monoclonal.

In principle, an organism is capable of producing, by itself, the so-called "drug" or antibody, by virtue of its immune system. However, the number of transformed cells circulating in the bloodstream, and thus the corresponding number of antigens, is generally very small in the early stages, which is important for diagnosis and therapy, and it is not possible to elicit a sufficient immune response. On the other hand, the method according to the invention provides large amounts of tumor-specific antigens in the form of transformed cell cultures and allows them to replicate as desired.

In order to separate the blood into its components, a device suitable for this purpose, for example the so-called apherese p
use the one used for rocess). In contrast to the conventional separation method, in which a large amount of whole blood is taken from a patient and separated into fractions using a centrifugation method, in the phlebotomy method, a separation device is connected by a line directly to the patient's bloodstream. There is. Thus, this method is in principle rather similar to the dialysis method.
In this way it is possible to separate only the desired fraction from the blood drawn from the patient and then immediately return other blood components to the patient. Therefore, the phlebotomy method enables a large amount of a predetermined blood fraction to be collected semi-continuously without burdening the patient. Therefore, the phlebotomy of plasma is called "plasmapheresis" and the collection of leukocytes is called "leukaphere".
sis) ”and the like (US Pat. No. 5,112,298,
U.S. Pat. No. 5,147,290).

To date, the only phlebotomy method known per se is to separate white blood cells, which in each case constantly circulate to the periphery in the circulatory system, from other blood components which are present in relatively large amounts. It has been considered suitable. Now, the present inventor has unexpectedly found that a large number of transformed cells extracted from a primary tumor are also contained in a specific cell fraction.

Separation of blood components is accomplished by centrifuge with a rotating vessel, such as is commonly used for phlebotomically separating blood plasma from red blood cells and other blood components without chemical density gradients and subsequent concentration. Machine (eg Haemonet in the US
It is advantageous to carry out in Haemonetics V50-1, commercially available from ics, so-called "Latham-Bowl"). The centrifuge itself has a blood volume of about 100-200 ml.

Heretofore, such a centrifuge has been, for example,
It has been used in combination with extracorporeal radiation therapy in a method of reducing lymphocyte populations in patients with lymphocytic leukemia (European Patent Publication No. 0 030 358). For this purpose, blood is taken from a patient and the blood is exposed to UV radiation in the presence of a photoreactive compound, such as a psoralen derivative, previously administered orally or after extraction to the blood. After psoralen intercalates with the DNA of cells and is photoactivated, DN
It can react with A, thereby eliminating the oncogene. The radiated blood is then returned to the patient.

The method of the invention comprises enriched transformed cells and leukocytes and / or leukocytes which are still contained under certain circumstances.
Alternatively, only the fraction with lymphocytes in it is protectively separated from the blood, while allowing other blood components to be returned to the patient. This is preferably done continuously in a single process. In this way, it is possible to keep the stress on the patient as low as possible, while isolating sufficient quantities of the transformed cancer cells in a short time and further processing such cells.

Prior to supplying blood, a centrifuge can be filled with a pharmaceutically acceptable isotonic solution to which a coagulation inhibitor such as heparin has been added. Usually 1,000-
2,000 units are sufficient for this purpose. To isolate transformed cells from the bloodstream via a suitable bypass mechanism attached to the outlet of the centrifuge, any number of white blood cells
Extract with the transformed cells mixed in them.
A person skilled in the art can easily identify the white blood cell fraction.

When a white band clearly distinguished from red blood cells appears, the centrifuge pie bath is opened and about 40 ml of white blood cells are taken out to the case of a centrifuge having a volume of 120 ml. It then returns red blood cells, plasma and other blood components back to the body.

The fraction enriched with transformed cells and, under certain circumstances, also leukocytes and / or lymphocytes (hereinafter simply referred to as "cell fraction") is transferred to the culture solution. For culturing transformed cells, each basic cell medium containing carbon and nitrogen sources and other necessary inorganic and organic constituents such as vitamins, amino acids and trace elements can be used. Commercially available media such as RPMI1
It is preferable to optionally add an additive such as fetal bovine serum to the 640 medium. Culture conditions are adjusted according to the manufacturer's information. Usually standard conditions of 5% CO ₂ , 37 ° C. and 95% humidity are used.

In the case of small primary tumors, which have probably not yet been diagnosed and localized or are in the pre-metastatic stage, only very small numbers of transformed cells circulate in the blood. Addition of macrophage inhibitory peptides to the cell fraction can prevent the macrophages from destroying the few transformants in a far excess of lymphocytes. In addition, addition of B-cell and T-cell specific antibodies (CD-8, CD-25, interleukin and interferon specific antibodies) suppresses lymphocyte production and finally these Can be killed. Alternatively, white blood cells, lymphocytes and other abnormal cells can be separated from the cell fraction by known cell sorting methods. Dispersing the batch into the wells of a microtiter plate reveals clearly visible clones of transformed cells after about 4 days. Transformed cells can be distinguished on the basis of their morphology from lymphocytes that are probably still present and are very similar to cells of the primary tumor. After about a week, sufficient numbers of transformed cells are obtained for subsequent immunization. Such clones can also be stored frozen for later use.

Cancer cells can be returned to the body as an immunogen in a modified, non-pathogenic form. For this purpose, for example, an isolated membrane fraction which still contains the tumor antigen can be used. For this, another foreign organism,
For example, mice, rabbits or goats, or the same organism from which the antigen was extracted is immunized with these antigens. Polyclonal or monoclonal antibodies produced by conventional methods can be used for diagnosis or therapy.

According to a preferred embodiment of the present invention, cultured cancer cells are used to stimulate B lymphocytes isolated from the same organism. For this purpose, it is advantageous to utilize the so-called in vitro immunization, ie the method of contacting B lymphocytes with transformed cells in a suitable medium in vitro. A hybridoma cell clone capable of permanently secreting the antibody is produced from the B lymphocytes thus stimulated. Apart from the hybridoma technology, it is also possible to immortalize B lymphocytes with Epstein-Barr virus. Thus, the present invention mimics the body's own immune system in a technically controllable manner.

In an embodiment of the invention, the transformed and expanded cells are cultured and expanded, at a given time point, preferably as soon as possible after removal, ie about 8 to 40 days later, in B lymphocytes resident in the patient. Contact with. Since isolation of B lymphocytes requires only about 50 ml of blood,
B lymphocytes can be centrifuged by density gradient, eg Ficoll
It can be isolated from whole blood in a gradient. But,
The extraction of B lymphocytes is advantageously carried out using a separation device used as lymphocyte phlebotomy according to the invention. Particularly preferably, both stages of treatment, ie application of the culture of cancer cells and isolation of B lymphocytes, are coordinated with one another so that the in vitro immunization is as early as possible after cell extraction, ie about 8-10. Be able to do it after a day.

Since only stimulated B lymphocytes secrete antibodies, the whole cell fraction (T lymphocytes and B lymphocytes) can be used for in vitro immunization. Here, the cell fraction and the transformed cells are preferably used in a ratio of 100: 1 to 1: 1 and particularly preferably about 10: 1 based on the number of cells. Interleukin,
It is preferred to stimulate the antibody forming cells, for example by the addition of IL-2, IL-4, IL-5, IL-6 and gamma interferon. The concentration of these adjuvants in the immunization batch was 0.1-10 mM each,
It is preferably about 1 mM.

In order to proliferate indefinitely, the stimulated B lymphocytes must be immortalized. This can preferably be carried out by transforming B lymphocytes with Epstein-Barr virus, similar to the method described in German Patent DE 39 28 769 C2. In order to produce a large amount of antibody, it is preferable to produce a hybridoma culture. For fusion with B cells, species-specific myeloma cells are used, depending on the donor organism (organism). For humans, the myeloma cell line U-266 has been found to be particularly suitable for this purpose. B cells are combined with myeloma cells in an optimal ratio of 1: 1. Additives such as polyethylene glycol can be added to support cell fusion. The batch is then sprinkled into the wells of a microtiter plate to select hybridoma clones and the culture supernatant is tested for antibodies. The culture conditions are those normally used for hybridoma culture.

By epitope selection, hybridoma clones recognizing various antigens, ie, determinants, on the surface of transformed cells are selected to prove the specificity of the antibody. The hybridoma cell line thus obtained can be further replicated in preparation for extraction of the antibody, or can be frozen and stored for later use.

The antibody is isolated from the culture supernatant by conventional methods and purified to homogeneity as far as possible by conventional protein purification methods such as ammonium sulfate precipitation, gel chromatography, ion exchange chromatography, affinity chromatography and / or FPLC and electrophoresis. Purify by.

In vitro immunization is carried out without reaching out to foreign organisms and supplies large amounts of antibody. These antibodies are specific to the particular tumor of the donor individual and are therefore optimal in this particular individual for use in their diagnosis and response. On the other hand, antibodies are obtained from B lymphocytes taken from the same individual.
Thus, although produced in vitro, these antibodies are "endogenous" proteins and do not elicit any immune response against themselves after being applied to the organism. If an immune response is elicited, it will lead to rapid degradation of these antibodies and thus diminished therapeutic activity.

The antibody produced by the method of the present invention is
All subclasses, eg IgG, IgM and Ig
Although it contains E, IgG type antibodies are preferred. Antibodies can be further modified chemically or enzymatically so long as their physiochemical activity, ie recognition and binding at antigenic determinants specific for cancer cells, remains.

Immunotoxins such as ricin or diphtheria toxin can be attached to the antibody by the conventional glutaraldehyde method. Furthermore, the antibody may be loaded with a radioactive substance, such as ¹²⁵ , in preparation for RIA or ELISA.
It can be labeled with I or a labeling enzyme.

In a preferred embodiment, the antibody is used to make the drug without further modification. At this time, the pharmaceutical composition may include one or more types of monoclonal antibodies directed against a particular epitope.

After being administered to a patient, such antibodies bind to attached tumor cells and thus can lure macrophages or endogenous killer cells, which play a role in cancer cells. Destroy.

The antibody is administered in a pharmaceutically acceptable formulation with conventional carriers, diluents, stabilizers and optionally other auxiliary substances. The drug can be present in a suitable formulation, preferably in the form of a solution that can be injected intravenously, intramuscularly or subcutaneously. Particularly preferably, the antibody is lyophilized after extraction, filled into single doses and sealed. Immediately before use, the antibody is dissolved or suspended in a carrier solution suitable for injection. The carrier solution, which is usually a sterile buffered aqueous solution, can be enclosed in its own ampoule with the lyophilized antibody in drug packaging. The physician will select the appropriate dose and mode of administration depending on the age and condition of the patient, the type of tumor and the severity of the condition.

In order to produce a drug corresponding to the tumor, the properties of the antibody can preferably be used to recognize and bind to its corresponding antigen with high specificity. The active ingredient, which was eutectic bound to the antibody in a suitable manner, i.e., except for the area important for antigen recognition, was directly carried to the desired position via the antibody as an excipient, and the activity was expressed only there. , And can be prevented from being dispersed unspecifically in the organism. Tumor cells can be destroyed by linking immunotoxins such as ricin or radiotherapeutic agents to these monoclonal antibodies by methods known per se.

By administering these modified antibodies and / or unmodified antibodies, tumor cells attached to endothelium can be detected and destroyed. Moreover, this method also allows the destruction of possibly non-surgical partially solid or solid tumors, eg metastases already formed. Following surgery, the antibody can be administered prophylactically over a corresponding period of time in order to prevent re-establishment of circulating transformed cells and development of metastases. Administration can also be carried out in conjunction with other conventional agents for the treatment of tumors, such as cytostatics, and associated therapeutic means, such as irradiation.

For diagnostic purposes, these antibodies can be linked to radioactive or non-radioactive labels and used in vivo and in vitro for the screening and labeling of tumor cells. The high selectivity and affinity of the antibody allows, by simple means, a quantitative and qualitative determination of the number of transformed cells in the patient's bloodstream in an in vitro test system. Thanks to the sensitivity of such tests, which can prove even a small number of cells in a sample, only a small amount of blood has to be drawn from the patient and the constant availability of antibodies allows for long-term testing. Can be carried out regularly to prepare for diagnosis.

An important feature of the present invention is that the products extracted according to the method, such as cultures of transformed cells, hybridoma cultures and antibodies are highly specific to the organism in question. This means that antibodies are somewhat more or less strong in their structure, especially with regard to their non-antigen recognition areas, from different organisms, eg different patients with the same tumor lesion. Antibodies, on the other hand, are actually endogenous to each individual and are not recognized as "foreign" by the immune system. The method according to the invention provides a drug or diagnostic tailored to a particular patient, for example by utilizing an antibody specific for that individual, in a relatively short time and at an acceptable cost. Allows to manufacture medicines. Such drugs, apart from their specific activity, are also characterized by high compatibility.

However, this does not mean that the use of antibodies produced for a particular individual from that individual is in principle excluded for other individuals. It is known that human antibodies of the same species are more compatible than chimeric or animal antibodies. If the antibody is used for in vitro diagnosis, compatibility plays no role anyway. Therefore, in the case of a patient suspected of having a specific tumor lesion or already diagnosed with such a medical condition, first experimentally,
An antibody obtained from another patient is used. If such an antibody reacts in the desired manner and is compatible, then as described above, it is no longer necessary to produce one's own antibody for this patient.

Nonetheless, if the type of primary tumor is known, in many cases B lymphocytes endogenous to the patient will be used using the method of the present invention, even though incompatible reactions may result. It is then sufficient to stimulate with a culture of transformed cells of the same type of cancer that have already been isolated and replicated from other individuals. Antibodies produced in this way are highly compatible as they are "quasi-endogenous" to the body. Only if such an antibody does not have sufficient specificity for the individual patient's cancer will it be necessary to carry out a complete isolation, culturing and immunization treatment by the method of the invention.

The present invention thus comprises a categorized and individually adaptable method for the manufacture of a medicament that can be used for the diagnosis and / or treatment of a particular cancer in a plurality of individuals of a particular mammal. To do.

In a preferred embodiment of the method of the present invention, a single clone is prepared which comprises different hybridoma cell lines obtained from one or several patients with the same tumor and which discriminate against specific antigenic determinants against cancer cells. Select according to sex antibody. In this way, it can be determined whether the same determinants occur for transformed cells isolated from different patients with the same tumor lesion. And
These antibodies can be used for diagnosis and possibly treatment in the case of other patients, without first performing immunization with endogenous B lymphocytes.

The antibody may be incorporated into the pharmaceutical composition in a modified or unmodified form by conventional methods, as described above.

[0046]

The simple, rapid and economical practicality of the method allows its use in the examination or early detection of cancer, even if the tumor has not yet been diagnosed. In addition, this is a test to examine the suspicion of cancer in the periodic diagnosis for the presence of transformed cells, and if there is a solid organ tumor that can be recognized by X-ray, the tumor is already in the circulatory system. It is even better suited to determine if the malignant cells have dispersed. Furthermore, if there are suspicious findings (transformed or non-transformed cells), tests can be performed for the presence of transformed cells circulating in the periphery. To this end, the antibodies and / or their particularly suitable derivatives can be utilized, preferably in a diagnostic kit for ELISA.

The above method can be applied not only in the field of human medicine but also in veterinary medicine to mammals in general and to related scientific fields. Therefore, what is described in the present specification by way of example of a human patient as a representative of a living individual should not be construed as limiting.

According to the method of the present invention, transformed cells can be extracted safely and without the addition of chemicals or other adjuvants from living organisms such as humans and animals for diagnostic and therapeutic purposes. . The method can extract large numbers of tumor cells circulating in the system. This can reduce tumor stress, which normally contributes to the metastatic process.

The method of the invention offers the following advantages, among others:

The extraction of transformed cells can be carried out very protective to the organism. That is, it does not increase the release of such cells into the bloodstream.

The present isolation method can very rapidly reduce the number of peripherally transformed cells in the bloodstream, and
For example, after surgery to remove the primary tumor, the risk of metastases forming is reduced.

By optimally matching the individual processing steps, polyclonal or monoclonal antibodies can be produced in a relatively short time and at low cost.

The extracted antibodies are individual-specific. That is, such antibodies are not foreign to the organism in question. There is no incompatibility and no excessive premature degradation of the antibody, in contrast to other foreign proteins. This substantially increases their efficiency.

Antibodies are tumor specific. That is, such antibodies recognize with high selectivity antigens specific to a particular tumor obtained from the subject individual. However, it does not exclude the antigen recognition of transformed cells obtained from other individuals.

The pharmaceutically active ingredients associated with the antibodies disperse their activity substantially only where desired (cellular targeting), rather than unspecifically dispersed in the organism. Therefore, undesirable side effects can be largely avoided.

The agents produced from the antibodies can be used not only for transformed cells circulating in the bloodstream, but also for primary tumors and possibly metastases already formed.

Drugs made from antibodies can be extracted from endogenous qualities while excluding foreign infections, eg by hepatitis or AIDS.

The extraction of tumor-specific antibodies for individual patients can be carried out at an acceptable cost, so that
In addition to producing these patient-specific drugs,
It can also be used for early detection, diagnosis or post-treatment.

Antibodies extracted from individual organisms can be used in organisms other than the "producing organism".

[0060]

The method of the present invention will be described in detail by the following examples.

Materials and Methods 1. Equipment used 1.1 Cell culture sterile workbench, CO ₂ incubator, incubator, heating plate, fluorescence microscope, phase contrast microscope, variable rotor centrifuge, cooler (4, -20, -80 ° C), liquid nitrogen, autoclave 1.2 ELISA microplate photometer (photometer), pipette,
8 channels 1.3 Purification of monoclonal antibody FPLC, HPLC 1.4 Biochemical analysis IEF equipment, midget system, electroblot, two-dimensional gel electrophoresis, transducer 1.5 Phlebotomy device Haemonetics V50-1 (Latham-bowl), capacity 120 ml, with bypass, made by Haemonetics, USA

2. Reagent 2.1 Tissue culture medium RPMI1640, fetal bovine serum, L-glutamine Na pyruvate, phosphate buffer solution (PBS), pH 7.
0, 20 mM, HAT medium 2.2 Monoclonal antibody anti-CD8, anti-CD25, anti-IL-2, anti-IFN-gamma (all obtained from Serva) Recombinant IL-2, IL-4, IL-5, IL -6, IF
N gamma (all obtained from Serva) Macrophage inhibitory peptide (Thr-Lys-Pro, tuftsin fragments 1 to 3) (obtained from Sigma) 2.3 50% polyethylene glycol in cell fusion HEPES buffer 15
00, 75 mM (50% weight / volume)

3. Method 3.1 Isolation of Cancer Cells by Phlebotomy and Using Haemonetics V50 Centrifuge with Replication Bypass,
Two patients with different types of tumors (prostate cancer, melanoma), each with blood 1 according to the method described above
Cell fractions of 20 ml each were extracted.

The histological stages of both patients were known from previous surgery.

2.4 · 10 ⁹ white blood cells obtained from the cell fraction were put into a tube together with the cell culture medium (RPMI1640), and examined as follows.

Standard conditions (37 ° C., CO ₂ 5%, humidity 95
%) And the cells were cultured overnight. As a medium, BMI and T cell specific antibodies (CD8, CD25, IL-2) to RPMI1640 with 10% fetal bovine serum were used.
And antibodies specific for IFN gamma) and macrophage inhibitory peptides were added at the following concentrations.

[0067] Anti-IFN 100 μg / medium 500 ml Anti-CD25 100 μg / medium 500 ml Anti-CD8 1mg / medium 500ml Macrophage inhibitory peptide (MIP) 50 mg / medium 500 ml

The batch was distributed on microtiter plates and plated with 200,000 cells in 100 μl per well. On the 4th day from the start of the culture, each cell was fed with 100 μl of medium (without addition of antibody or MIP additive). CO ₂ 5% at 37 ° C. throughout the culture period
Constantly maintained. After 4 days, the cancer cells after division were clearly visible as clones in a phase contrast microscope.

For comparison purposes, the same batch was plated in medium without the addition of antibody and macrophage inhibitory peptide. Only after 3 weeks, the cancer cells could be confirmed as clones by a phase contrast microscope. Batches of clones with antibody and macrophage inhibitory peptide added were picked and replicated until 10 ⁸ cells were obtained.
Additional clones were duplicated and stored frozen.

3.2 Immunizations Leukocytes had to be secreted to secrete antibodies against cancer cell-specific antigens. For this purpose, in one of the phlebotomy cycles, 10 ⁹ white blood cells were taken out, combined with 10 ⁸ cancer cells and 4 times in 100 ml human plasma supplemented with heparin.
The cells were cultivated at 37 ° C. and CO ₂ 5% for a day. IL-
The addition of 2, IL-4, IL-5, IL-6 and IFNgamma promoted maturation of antibody forming cells. The concentration is 10 per 100 ml of immunization batch.
Reached 0 μg.

3.3 Cell fusion B lymphocytes were separated from other cells by FACS.
Several days before the fusion, human myeloma cells (U266) were maintained at a density of 3.10 ⁵ cells / ml during the logarithmic growth period. Myeloma cells are harvested at the same time as B lymphocytes and P
Washed 3 times in BS. We prepared four fusion batches, respectively, myeloma cell 5 - 10 ⁷ B-lymphocytes 5 &
Centrifuge with 10 ⁷ and then HE
Fused with 1 ml of 50% polyethylene glycol in PES. Then slowly rotate RPMI 1640 to 10
ml was added. The fusion batch was washed in RPMI 1640, centrifuged at 200G for 2 minutes at room temperature and HAT
It was put in 50 ml of each medium, and 0.1 ml was spread per well in a 96 well type microtiter plate. 10 ⁴ human macrophages per well were used as feeder cells.

3.4 Screening Hybridoma cell culture supernatants were tested for antibodies by ELISA. A cell ELISA was prepared for this purpose. By appropriate methods, cancer cells were applied to the plastic side of microtiter plates and the antibody, possibly bound to those plates, was detected by a second antibody.

ELISA Immunoglobulin-secreting clones were sought in the first selection after diffusion.

1. Culture supernatants were incubated in microtiter plates at 100 μl per well overnight at 4 ° C.

2. Washed 3 times with PBS.

Blocked with albumin or blotto at 37 ° C for 3.2 hours.

4. Washed 3 times with PBS.

5. Antibodies specific for human immunoglobulin were incubated for 2 hours at 37 ° C.

6. The second antibody conjugated to the enzyme (peroxidase or alkaline phosphatase) was incubated for 1 hour 37
Cultured at ° C.

7. Express by adding substrate,
It was measured with a reader.

Clones positive for antibodies specific for cancer cells were tested. For this, a cell ELISA was prepared.

1. 1 mg of poly-L-lysine in 1 ml of distilled water
Was prepared.

2. Microtiter plates with poly-L-lysine were incubated for 15 minutes.

3. Washed with water. After drying, it was stored at room temperature.

4. Suspend cells in PBS and wash, 4
Centrifuged at 00G.

5. 10 cell pellets per ml of PBS
Resuspended in ⁵ pieces. Fill microtiter plates and incubate for 10 minutes at room temperature.

Fixed acetone / methanol mixture (50/50% by volume) 20
0 μl was poured into each well of the microtiter plate and incubated at room temperature for 2 minutes. Then, it wash | cleaned once with PBS.

The subsequent steps were carried out in the same way as the first selection.

3.5 Biochemical Analysis Monoclonal antibodies obtained directly from the culture supernatant were analyzed.
The L and H chain subclasses were determined in an ELISA by the Sucbrus test kit. Isoelectric focusing was used to confirm the monoclonality of the antibody (Hoffmann et al., Human Genetics, Springer Verl.
ag, 1990, 84, 137-146).

3.6 Epitope selection (screening) Cancer cells were cultured, 10 ⁷ cells were taken out, and 3
It was washed twice and divided into two fractions. One was homogenized and analyzed by negative gel electrophoresis (pH 8.8, 9% acrylamide in the separating gel, 3% acrylamide in the collecting gel), the other was denatured (urea, SDS), one-dimensional. And two-dimensional gel electrophoresis (Waldinger et al., Electorop
horesis, 1986). Subsequent Western blot detection was performed on the culture supernatant. The bands and / or spots found were extracted and sequenced, thus identifying tumor-specific antigens. Other tissues and white blood cell samples were used as controls (E. Harlowe, D. L.
ane, Antibodies, A Laboratory Manual, Cold Spring
Harbor Laboratory, 1988; D. Baron, U. Hartlaub, Hu
mane monoklonale Antikoerper, Gustav Fischer Verla
g, Stuttgart, New1987).

4. Results In any case (prostate cancer, melanoma), after 4 days, in about 30% of the wells of the cell culture plate, clones of the transformed cells could be confirmed by phase difference.

The cell fraction of patients with non-malignant lesions (dysfunctional autoimmune regulation of lymphocytes) was used as a comparative control in which leukocytes had to be treated in vitro for other reasons. In this case, no cell division was seen.

In the same manner, the following transformed cell lines were isolated, and hybridoma cultures secreting the corresponding antibody were produced from these lines.

Breast cancer B-cell lymphoma Bronchial cancer Renal adenocarcinoma

Deposition of Cancer Cells and Hybridoma Cell Lines The following cultures of transformed cells (cancer cell lines) were produced, and the corresponding hybridoma cell lines were produced from these cultures.

[0096]

[Table 1]

These hybridoma cell lines were stable and secreted 5-7 μg of antibody per 10 ⁶ cells during 24 hours (according to the literature, human hybridomas were:
The average value is 1 to 10 μg of antibody per 10 ⁶ cells in 24 hours).

Some of these cancer cell lines and hybridoma cell lines were obtained according to the Budapest Agreement of August 17, 1993, Deutsche Sammlung fuer Mikroorga.
nismen, DSM (Germany, Mascheroder Weg
1b, 38 124 Braunschweig).

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI A61K 39/395 A61P 35/00 A61P 35/00 C12N 5/00 E (72) Inventor Ulrich Kübler Germany, Day-80538 München, Lerchenfeldstraße 20 (72) Inventor Reinel Hoffmann, Federal Republic of Germany, Day 82256 Fürstenfeldbruck, Wettersteinplatz 1 (56) Bibliography WO 93/000120 (WO, A1) Br. J. Cancer, 1983, Vol. 48, p. 665-673 Biochem. J. , 1980, Vol. 191, p. 239-246 Biochemical Pharmacology, 1981, Vol. 30, No. 24, p. 3333-3336 Cancer and Chemotherapy, 1988, Vol. 15, No. 5, p. 1677-1684 (58) Fields investigated (Int.Cl. ⁷ , DB name) C12N 5/00-5/28 C12P 21/08 G01N 33/53 A61B 10/00 BIOSIS / WPI (DIALOG) PubMed

Claims (13)

(57) [Claims] 1. A method for in vitro isolation of transformed cells, which comprises preparing a collected blood sample and using a phlebotomy device to obtain a blood fraction containing leukocytes and / or lymphocytes. A method comprising the step of separating from other blood components, wherein the cell fraction contains a large number of transformed cells (however, in the above method, the step of extracting blood from the individual and other blood components to the individual Is not included). 2. The method of claim 1, wherein the blood sample is whole blood. 3. The method further comprises the step of replicating transformed cells in the presence of at least one of (1) macrophage inhibitory peptide; (2) B lymphocyte-specific antibody and (3) T lymphocyte-specific antibody. The method according to claim 1 or 2. 4. The transformed cells are (1) breast cancer and (2) B.
Cell lymphoma, (3) renal adenocarcinoma, (4) bronchial cancer, (5)
The method according to any one of claims 1 to 3, which is selected from the group consisting of prostate cancer and (6) melanoma. 5. The method of any one of claims 1 to 4, further comprising replicating the transformed cells in vitro. 6. A method for detecting the presence of transformed cells in blood, comprising the steps of preparing a collected blood sample; using a phlebotomy device to fractionate blood containing leukocytes and / or lymphocytes. And a step of separating a cell fraction containing a large number of transformed cells from other blood components (however, the above steps include a step of extracting blood from an individual and a step of returning other blood components to the individual. No); and (i) culturing the blood fraction and allowing the transformed cells present in the fraction to grow and be detected, and / or (ii) the fraction being specific for a tumor-specific antigen. A method comprising screening the blood fraction for the presence of transformed cells by contacting the antibody with a different antibody and confirming the presence of transformed cells in the blood fraction. 7. The method according to claim 6, wherein the antibody is a monoclonal antibody. 8. The method of claim 6, wherein the antibody is a polyclonal antibody. 9. A method for producing an individual-specific antibody, which comprises culturing the transformed cells obtained by the method according to any one of claims 1 to 5 together with a fraction containing leukocytes and / or lymphocytes. , Inducing an individual-specific immune response in vitro. 10. The method according to claim 9, wherein the transformed cells are contacted with the fraction containing leukocytes and / or lymphocytes after culturing the transformed cells for a certain period of time. 11. The method according to claim 9 or 10 for preparing an individual-specific antibody against a tumor-specific antigen. 12. The method according to claim 11, wherein the B lymphocyte is immortalized. 13. The method according to any one of claims 9 to 12, wherein the fraction containing leukocytes and / or lymphocytes is derived from the same individual from which the transformed cell is derived.